Inflammatory and coagulation biomarkers linked to mortality in large treatment interruption trial
Richard Jefferys, TAG
The strong association between immune activation and disease progression in HIV infection has been demonstrated and confirmed by multiple studies over the past 25 years.
In several reports, expression of the CD38 activation marker by CD8 T cells has correlated more closely with progression than viral load or CD4 T cell counts. Elevated levels of immune activation were even noted in the first AIDS case reports published in December 1981 in the New England Journal of Medicine (NEJM) ; at that time, CD38 was known as the thymocyte-associated antigen T10.
Research has also shown that immune activation significantly increases the levels of pro-inflammatory cytokines in people with HIV infection compared to uninfected controls. Suppression of HIV replication by antiretroviral therapy (ART) typically causes an immediate and precipitous decline in immune activation, but levels tend to remain slightly higher than in controls even after several years of treatment.
What has been relatively unclear until recently is the degree to which the immune activation and inflammation caused by HIV infection can impact health. This under-appreciated aspect of disease pathogenesis was highlighted by the outcome of a large, 5,472-person randomised trial known as SMART (Strategies for the Management of AntiRetroviral Therapy), which compared continuous ART with an intermittent, CD4-guided ART strategy. 
Smaller studies had suggested this approach might be a safe and potentially toxicity- and cost-sparing approach to treating HIV and the primary risks were thought to involve drug resistance, increased possibility of transmission and acute infection symptoms after interruption. Only a few clinicians, such as Diane Havlir, voiced concern that the inflammatory consequences of HIV replication might cause treatment interruptions to be harmful. It was thus a surprise to many when in late 2006, it was announced that the intermittent therapy arm of SMART was being halted due to a doubling of the risk of illness and death compared to the continuous ART arm.
The results, published subsequently in NEJM, showed that the intermittent arm did worse on every endpoint, and the increased risk of clinical events and death was seen across all CD4 strata, not just among individuals with low baseline counts or nadirs. The majority of the events involved cardiovascular, liver and kidney disease and not opportunistic infections, suggesting that immune activation and inflammation had played a larger role in the outcomes than immunodeficiency. 
Now, in the latest PLoS Medicine, the SMART researchers report that analyses of biomarkers of inflammation and blood coagulation offer strong evidence that this was indeed the case. 
The study authors begin by noting that prior studies have demonstrated links between HIV replication and endothelial cell dysfunction (endothelial cells line the interior surface of blood vessels), and that this was associated with elevations in markers of hypercoagulability (a tendency for the blood to clot inappropriately) which were reduced by ART. Levels of the pro-inflammatory cytokine IL-6 have also been shown to be elevated in cross-sectional studies of HIV-infected individuals compared to uninfected controls, and IL-6 levels correlate with viral load in advanced disease. Similarly, levels of the pro-inflammatory marker C-reactive protein have been shown to increase over the course of HIV infection, with the highest levels reported among individuals progressing to AIDS.
In the SMART trial, a pre-planned substudy regularly measured four inflammatory biomarkers: high sensitivity C-reactive protein (hsCRP), IL-6, amyloid A, and amyloid P, and two coagulation markers: D-dimer and prothrombin fragment 1+2. To evaluate the impact of these biomarkers on the risk of death among SMART participants, the researchers conducted a case-control study in which two controls were matched with each of the 85 participants who died during the trial in order to compare biomarker levels using the samples taken prior to death. A second analysis was also conducted to look at levels in a larger random sample of 250 participants from each trial arm. Additional analyses looking at links between these biomarkers and specific clinical events are also being conducted, but results will be published separately.
The results showed that IL-6, D-Dimer and C-reactive protein levels were significantly higher among individuals that died compared to the matched controls.
In the larger analysis of a random sample of 250 participants from each arm after one month on study, the researchers found that individuals randomized to interrupt ART showed significant increases in IL-6 and D-Dimer levels: the median increase in IL-6 was 0.60 pg/ml (a 30% increase) and for D-Dimer 0.05 ug/ml (a 16% increase). In comparison, the continuous ART group showed median increases of 0.0 ug/ml and 0.12 pg/ml (a 5% increase) for D-dimer and IL-6, respectively. The researchers report that changes in hsCRP and amyloid A were in the same direction but did not differ significantly between the two groups. The largest increases were seen among individuals in the interruption group who entered the study on ART with viral loads below 400 copies/mL (a 27% increase in D-Dimer and 43% increase in IL-6).
In discussing their results, the authors note that elevated IL-6 and D-Dimer levels have been associated with all-cause mortality in studies of uninfected elderly and critically ill individuals, lending plausibility to the associations observed in SMART. [4, 5]
They also point out that while inflammation is the most likely factor driving these biomarker elevations, the study does not formally prove this, and other potential contributors could involve microbial translocation (which elevates LPS, which in turn can elevate D-Dimer and provoke IL-6 production by monocytes) and direct effects of HIV proteins or HIV replication. One implication of the data highlighted by the researchers is that “specific therapies that reduce the inflammatory response to HIV and decrease hsCRP, IL-6, and D-dimer levels may warrant investigation as an approach for reducing risk of death among HIV-infected individuals.”
A potentially important and worrying issue raised by these results which isn’t discussed in the paper is that studies of treatment interruptions are likely more dangerous to people’s health than has previously been appreciated. The general consensus has been that restricting this type of research to individuals with high CD4 counts and regularly monitoring CD4 counts and viral load during ART interruption is sufficient to ensure safety. These new data indicate that, at the very least, IL-6 and D-Dimer levels should be evaluated in order to exclude those at highest risk from treatment interruption trials. Clear information about these findings also needs to be added to the informed consent of any ART interruption protocol.
One example is a recently initiated trial of a therapeutic vaccine made by Bionor Immuno. 
This study involves a six month ART interruption and is enrolling people with CD4 counts over 400 and nadirs over 200. The SMART data show that, without additional safeguards, this type of trial is likely to put participants at unnecessary risk of illness and death.
TAG Basic Science Blog (22 October 2008).
- Gottlieb MS et al. Pneumocystis carinii pneumonia and mucosal candidiasis in previously healthy homosexual men: evidence of a new acquired cellular immunodeficiency. NEJM Volume 305:1425-1431. (10 December 1981)
- SMART Study Group. CD4+ Count-Guided Interruption of Antiretroviral Treatment. NEJM Volume 355:2283-2296. (30 November 2006)
- Kuller LH et al for the INSIGHT SMART Study Group. Inflammatory and Coagulation Biomarkers and Mortality in Patients with HIV Infection. PLoS Medicine Vol. 5, No. 10, e203 doi:10.1371/journal.pmed.0050203. Free access to full text
- Cohen HJ et al. Coagulation and activation of inflammatory pathways in the development of functional decline and mortality in the elderly. Am J. Med. 2003 Feb 15;114(3):180-7.
- Shorr AF et al. D-dimer correlates with proinflammatory cytokine levels and outcomes in critically ill patients. Chest 2002;121:1262-1268.
- Immunogenicity study of Vacc-4x versus placebo in patients infected with HIV. Due to enrol 345 patients from August 2008.